This present invention relates to methods, tissue culture medium, and kits for accelerating the production living tissue equivalents.
Recently, various systems for the in vitro production of tissue equivalents have been described. As used herein, the term xe2x80x9ctissuexe2x80x9d comprises any group or layer of cells which together perform one or more certain functions. Such tissue equivalents include, but are not limited to, equivalents of epithelial tissue, connective tissue, cartilage, bone, organs, vascular grafts, glands and blood vessels and comprise living cells and extracellular matrix molecules, principally collagen, and may optionally be provided with components not typically found in normal tissue.
Three-dimensional cell culture systems have been described which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time (U.S. Pat. Nos. 5,624,840; 5,541,107; 5,521,087; 5,516,681, 5,516,680; 5,512,475, herein incorporated by reference in their entirety). Cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts, actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.
These inventions are based, in part, on the discovery that growth of stromal cells in three dimensions will sustain active proliferation of cells in culture for longer periods of time than will monolayer systems (U.S. Pat. No. 5,510,254). This may be due, in part, to the increased surface area of the three-dimensional matrix which results in a prolonged period of active proliferation of stromal cells. These proliferating stromal cells elaborate proteins, growth factors and regulatory factors necessary to support the long term proliferation of both stromal and tissue-specific cells inoculated onto the stromal matrix. In addition, the three-dimensionality of the matrix allows for a spatial distribution which more closely approximates conditions in vivo, thus allowing for the formation of microenvironments conducive to cellular maturation and migration. The growth of cells in the presence of this support may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and other materials to the support itself or by coating the support with these materials.
Similarly, tissue equivalents comprising a hydrated collagen lattice contracted by a contractile agent, such as fibroblast cells or blood platelets, in combination with a variety of cell types to form the tissue equivalent are disclosed in U.S. Pat. Nos. 4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; and 4,835,102; 4,837,379; 5,256,418; 5,536,656; and RE 35,399, all of which are incorporated herein by reference.
In specific embodiments of the invention, bone marrow, bone, skin, dermis, liver, kidney, cartilage, ligament, tendon, pancreas, and heart valve tissues may be grown in the three dimensional culture system. The resulting cultures have a variety of applications ranging from transplantation or implantation, in vivo, of cells grown in the cultures, cytotoxicity testing and screening compounds in vitro, and the design of xe2x80x9cbioreactorsxe2x80x9d for the production of biological materials in vitro. For example, skin tissue equivalents can be used as grafts to treat burn victims or ulcer patients, while kidney and liver tissue equivalents can be used for transplanting where disease has caused organ damage or failure. For diffuse tissues such as bone marrow, the proliferating cells could be isolated from the culture system for transplantation. Tendon, ligament, and cartilage tissue equivalents can be used for transplantation or prosthetics for seriously damaged tissue.
While the methods described above have met with some success, improved methods that accelerate the generation or improve the quality of tissue equivalents would be useful for more rapidly providing usable tissue equivalents. In particular, it would be useful to provide improved methods that promote more rapid acceleration of the cell type of interest and that accelerates the production of extracellular matrix by stromal cells in the tissue equivalent, and also improves the quality of the extracellular matrix produced.
The present invention provides methods that increase the production of tissue equivalents that are useful in transplantation therapy, drug testing, cytotoxicity testing of compounds, production of cellular compounds in quantity, and laboratory testing of tissue systems.
In one aspect, the present invention provides improved methods for accelerating the production of tissue equivalents by contacting the tissue equivalent with angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II ( AII), AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.
In another aspect of the present invention, an improved cell culture medium is provided for the production of tissue equivalents, wherein the improvement comprises addition to the culture medium of an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.
In a further aspect, the present invention provides kits for the production of tissue equivalents, wherein the kits comprise an effective amount of angiotensinogen, AI, AI analogues, and/or AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof, and/or AII AT2 type 2 receptor agonists, and instructions for culturing the tissue equivalents.